Raman spectroscopic microscopes are quite effective to observe biologically-related samples. In a Raman spectroscopic microscope, an observation target is irradiated with a focused laser beam to detect Raman scattered light generated from the observation target. Raman scattered light has a shifted frequency from the frequency of the excitation light, and a Raman spectrum is measured with a spectrometer or the like. Scanning an observation target with an irradiation beam while changing their relative positions can obtain an optical spectrum at each position, and an image can be formed on the basis of such spectrum. A Raman spectrum at each observation position reflects the vibrational excited state of a molecule at the position, and thus is characteristic of the molecule. Using the characteristics of such a spectrum can know, if living cells are observed, a distribution of biomolecules in the cell tissue.
FIG. 2 shows a process by which Raman scattering occurs, using an energy level diagram. Raman scattering includes Stokes scattering and anti-Stokes scattering. FIG. 2 shows only Stokes scattering. Reference numeral 701 denotes the molecular vibrational ground state, and reference numeral 702 denotes the vibrational excited state. When a molecule is irradiated with a pump beam with a frequency top, a beam with a frequency ωS is scattered after an intermediate state 703 is once reached. At this time, the molecule falls back to one of the levels of the vibrational excited state 702. The scattered beam with the frequency ωS is a Stokes beam with a frequency lower than that of the pump beam. The molecular vibrational excited state has a plurality of levels, and the vibrational excited state differs depending on the types of molecules. Further, as the probability of transition from the level of the intermediate state to the level of the vibrational excited state differs from molecule to molecule, a spectrum that is unique to the molecule is formed. The Raman shift frequency Ω is represented by Ω=ωP−ωS, and has a positive value in the case of Stokes scattering. In the case of an anti-Stokes beam, the initial state is the molecular vibrational excited state, and the molecular state falls back to the vibrational ground state after an intermediate level is once reached. In such a case, if the frequency of the anti-Stokes beam is represented by ωAS, ωP<ωAS. Thus, the frequency of the anti-Stokes Raman scattered beam is higher than that of the pump beam.
Measurement of the aforementioned Raman scattering takes a long time as the intensity of the obtained scattered light is weak. As a method that can obtain intense scattered light, there is known spectroscopy that uses nonlinear Raman scattering called CARS (Coherent Anti-Stokes Raman Scattering). Using such a method can also obtain a Raman spectrum and know the molecular vibrational state. To generate CARS, pulsed laser with high peak power is used. CARS is generated from such a pulsed laser beam due to the nonlinear effect, and the intensity of the CARS becomes orders of magnitude higher than that of Raman scattering as the peak power is higher. Accordingly, it is possible to obtain a signal with a high signal-noise ratio and significantly reduce the measurement time.
CARS is based on the third-order polarization. In order to generate CARS, a pump beam, a Stokes beam, and a probe beam are required. Typically, the pump beam is substituted for the probe beam in order to reduce the number of light sources. In that case, the induced third-order polarization is represented as follows.PAS(3)(ωAS)=|χr(3)(ωAS)+χnr(3)|EP2(ωP)E*S(ωS)  [Formula 1]
Herein, χr(3)(ωAS) is a resonant term of a vibration of a molecule with the third-order electric susceptibility, and χnr(3), which has no frequency dependence, is a nonresonant term. In addition, the electric fields of the pump beam and the probe beam are represented by EP, and the electric field of the Stokes beam is represented by ES. In Formula (1), the asterisk that appears in ES represents the complex conjugate. The intensity of a CARS beam is represented as follows.ICARS(ωAS)∝|PAS(3)(ωAS)|2  [Formula 2]
A mechanism by which a CARS beam is generated will be described using a molecular energy-level diagram (FIG. 3). FIG. 3 shows a process of the resonant term. As in FIG. 2, reference numeral 701 denotes the molecular vibrational ground state, and reference numeral 702 denotes the vibrational excited state. A molecule is simultaneously irradiated with a pump beam with a frequency ωP and a Stokes beam with a frequency ωS. At this time, the molecule is excited to a vibrational excitation level 702 after an intermediate state 703 is once reached. When the molecule in the excited state is irradiated with a probe beam with a frequency ωP, the molecule falls back to the vibrational ground state while generating a CARS beam with a frequency ωAS after an intermediate state 704 is once reached. The frequency of the CARS beam at this time is represented by ωAS=2·ωP−ωS.
FIG. 4 shows a process related to the nonresonant term in Formula (1). This is a process in which an intermediate state 705 is once reached but the frequency of the Stokes beam is not in the vibrational excited state. The intermediate state 705 in which electrons and the like are involved is excited when a molecule is simultaneously irradiated with a pump beam with a frequency ωP and a Stokes beam with a frequency ω′S. When the molecule is further irradiated with a probe beam with a frequency ωP, a nonresonant CARS beam with a frequency ωAS is generated after an intermediate state 704 is once reached. When a broadband laser beam is used as a Stokes beam, for example, it may contain a beam with a frequency ω′S in FIG. 4 and the like. Such resonant CARS beam and nonresonant CARS beam are coherent with each other and thus interfere with each other.
Since Raman scattering was first discovered in 1928, a spectrum of a variety of molecules has been researched, and data thereon has been accumulated. Thus, it is desirable to identify molecules with reference to such spectral data. A CARS beam is represented by Formulae (1) and (2), and Im[χr(3)(ωAS)] is a portion corresponding to the Raman scattering spectrum. This is the complex portion of the resonant term, and interferes with the nonresonant term χnr(3) as described above. Thus, the shape of the spectrum obtained from CARS differs from that of the Raman scattering spectrum Im[χr(3)(ωAS)]. Therefore, it would be difficult to directly analyze a CARS spectrum with reference to the Raman scattering spectrum.
Development of a method for extracting a Raman scattering spectrum from a CARS spectrum is an important challenge to be addressed, and a variety of methods has been developed (Non Patent Literature 1). For example, the maximum entropy method, which is a method for restoring a phase spectrum from an intensity spectrum, includes determining a complex portion of a resonant term through mathematical computation. Alternatively, a method that uses interference is also known (Non Patent Literature 2).
As a spectral region that is sensitive to the molecular structure, there is a Raman scattering spectral region (of from 1800 to 800 cm−1) called a fingerprint region. For detection of a CARS beam, a spectrum in a similar region is desirably obtained. In the method introduced in Non Patent Literature 1, the spectral bandwidth of a Stokes beam for excitation is about 140 cm−1, which cannot cover such region. Non Patent Literature 3 introduces a method that uses a photonic fiber for a light source to cover such deficiency. Specifically, the method includes irradiating a photonic fiber with ultrashort pulsed laser to generate a broadband beam called a supercontinuum beam, and using it as a Stokes beam.